Brush Border

This study evaluated the effects of replacing fish meal (FM) in the diet of European sea bass (Dicentrarchus labrax) with differently processed soybean meals on growth performance; apparent digestibility coefficients (ADCs); the activity of alkaline phosphatase and the brush border digestive enzymes: leucine amino peptidase, maltase and γ-glutamyl transpeptidase; and the fish morphometric and flesh quality. European sea bass with an initial average body weight of 187.8 ± 1.4 g, were fed either a FM based (control) diet or a soy replacement isoproteic, isolipidic and isocaloric diet. The treatments consisted replacing FM protein by: 25% toasted, dehulled and solvent-extracted soybean meal (SE25); 50% dehulled and toasted soy seeds subjected to dry extrusion and mechanical oil extraction (ME50); 50% enzyme-treated soybean meal (ET50) and an inclusion of 60% composed of 30% toasted, dehulled and solvent-extracted soybean meal and 30% enzyme-treated soybean meal (SE + ET/60). ADCs values of dry matter, crude protein, crude lipid and gross energy did not significantly differ between ME50 and SE + ET/60 but both of them were inferior to the other treatments (P < 0.05). Compared with the control FM-based diet, only the group that consumed the ME50 diet had lower specific growth rate, feed conversion ratio and gross protein retention efficiency values (P < 0.05). The protein efficiency ratio did not differ between the FM-based control group and all other soy derivate groups (P > 0.05). In order to gain more insight on the effects of the diet on fish growth, activity of the brush border enzymes was measured. No significant differences were found in leucine amino peptidase and maltase activities, between the control group and the other soy derivate groups. The activity of γglutamyl transpeptidase was significantly higher in the upper section in fish that consumed the control diet compared with fish that consumed the soy derivate diets. Yet, the main activity of this enzyme was found in the lower intestinal section. In all the soy derivate diets except for the SE25 diet, the activity of alkaline phosphatase was significantly lower than its activity in the control diet. Feeding diets that include various types and levels of soybean derivatives did not affect the whole body composition and slaughter yield but significantly reduced liver weight. The results show that in methionine supplemented diets, FM can be replaced in terms of protein by 25% SE, 50% ET or 60% soy composed of 30% SE and 30% ET without hampering fish performance. The inclusion of 50% protein from ME resulted in significantly lower growth performance and feed utilization.

The Asian sea bass is a carnivorous, euryhaline species originating in seawater. It is currently being reared under intensive conditions in freshwater re-circulating ponds. The effects of feeding juvenile Asian sea bass at different levels during the day and/or night on their growth performance and on the activity of the brush border enzymes were evaluated in closed, freshwater re-circulating tanks. Contrary to previous published data, the results of the present study showed that the feeding time factor had almost no affect on fish growth rate. Asian sea bass appear to consume food whenever it is fed, even during the night. However, the feeding ration significantly affected growth rate. Restricted feeding (2% of body mass per day) significantly reduced growth rate, while feeding in excess (6% of body mass per day) did not improve it compared with a daily ration of 4%. The feeding ration also had an impact on survival, which was significantly lower in the groups which were fed 2% of body mass (during either the day or night) than in the other groups. The coefficient of variance (CV) of weights was influenced by both the feeding level and the time of feeding. It increased as the feeding ration decreased and also when the ration was fed at night. Feeding 4% during both day and night resulted in a similar growth rate to that obtained with either day or night feeding at this level. The brush border enzyme activity was affected by both the feeding time and ration. It was higher in groups fed 2% of body mass and in groups fed during the day. The proteases were affected to a greater extent than the carbohydrases. The high activity of all the tested enzymes in the pyloric caeca points towards their major importance in food digestion and absorption in this species. D

Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of: (i) pepsin, (ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of ␤-lactoglobulin (␤-Lg), and especially that of the peptide ␤-Lg f125-135, could have implications for the immunogenic action of ␤-Lg in the insurgence of cow's milk allergy.

The aim of this study was to provide data on the morphology of the gastrointestinal tract of Adélie penguins (Pygoscelis adeliae). It was found to consist of a long oesophagus, a two-chambered stomach, a small intestine measuring only 5.2&#50body length, two rudimentary caeca and a short colon, typical of carnivorous birds. The stomach comprised a glandular proventriculus and a muscular

The family of attaching and effacing (A/E) bacterial pathogens, which includes diarrheagenic enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), remains a significant threat to human and animal health. These bacteria intimately attach to host intestinal cells, causing the effacement of brush border microvilli. The genes responsible for this phenotype are encoded in a pathogenicity island called the locus of enterocyte effacement (LEE). Citrobacter rodentium is the only known murine A/E pathogen and serves as a small animal model for EPEC and EHEC infections. Here we report the full DNA sequence of C. rodentium LEE and provide a comparative analysis with the published LEEs from EPEC, EHEC, and the rabbit diarrheagenic E. coli strain RDEC-1. Although C. rodentium LEE shows high similarities throughout the entire sequence and shares all 41 open reading frames with the LEE from EPEC, EHEC, and RDEC-1, it is unique in its location of the rorf1 and rorf2/espG genes and the presence of several insertion sequences (IS) and IS remnants. The LEE of EPEC and EHEC is inserted into the selC tRNA gene. In contrast, the Citrobacter LEE is flanked on one side by an operon encoding an ABC transport system, and an IS element and sequences homologous to Shigella plasmid R100 and EHEC pO157 flank the other. The presence of plasmid sequences next to C. rodentium LEE suggests that the prototype LEE resided on a horizontally transferable plasmid. Additional sequence analysis reveals that the 3-kb plasmid in C. rodentium is nearly identical to p9705 in EHEC O157:H7, suggesting that horizontal plasmid transfer among A/E pathogens has occurred. Our results indicate that the LEE has been acquired by C. rodentium and A/E E. coli strains independently during evolution.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3 region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5 end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3 region of this new intimin type. This intimin, referred to as "," was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin ␤, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins ␣ and ␥.

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzymelabeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased ϳ10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4 -6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixedlipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP Ͼ10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5Ј-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125 I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125 I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125 I-Cry1Ab binding to BBMV. Cry1F inhibited 125 I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

An important early step in the colonization of the human gastrointestinal tract by bacteria is the adhesion of the organism to the host surface. Although adhesion is essential to maintain members of the normal microflora in the intestine, it is also the critical early phase in all diarrheal infections caused by pathogenic Escherichia coli strains. It is important, therefore, to fully understand the mechanisms underlying E. coli adhesion and in that way to be able to develop methods of maintaining the intestinal normal microflora and to prevent pathogenic E. coli from initiating an infectious process. Great progress has been made in recent years in the identification of the adherence factors of different diarrheagenic E. coli strains (Table 1). These protein structures are associated with the bacterial surface and can be subdivided into fimbrial and nonfimbrial adhesins (Fig. 1). In this minireview, we will discuss recent advances in the identification and characterization of previously known and novel adhesion factors from the six major categories of diarrheagenic E. coli strains: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adhering E. coli (DAEC).

The isolation of viable enterocytes, villi and crypts from the small intestine of a feral bird (Columba livia) is important for performing physiological experiments in ecologically relevant processes of membrane transport. The effectiveness of mechanical disruption, enzymatic digestion and chelating agents were compared. The objectives were to isolate enterocytes, villi and crypts from the small intestine of young pigeons; to evaluate the viability of the isolated intestinal epithelial cells isolated; and to verify the integrity of enterocytes by biochemical features. Enzymatic and mechanical methods yielded both elongated columnar and spherical cells. With the chelating method villi and crypts were obtained. All methods produced a high yield of intestinal epithelial cells with about 50 % viability. Brush border enzymes (sucrase-isomaltase and alkaline phosphatase) activities were high and, as reported in chickens, they did not differ along the intestinal villus-crypt axis. Although the three methods have good viabilities, the enzymatic technique gives the best yield in cell number, while the chelating method provides the highest populations of morphologically distinctive villi and crypts.

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 Å) and in complex with acarbose (to 1.9 Å). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

Background The brush-border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and detoxifies different toll-like receptor ligands. This study aimed to determine the therapeutic effects of locally administered calf IAP (cIAP) in a cecal ligation and puncture (CLP) model of polymicrobial sepsis. Methods C57BL/6 mice underwent CLP followed by intraperitoneal injection of cIAP or normal saline. Blood leukocyte counts, levels of cytokines and liver enzymes, and lung myeloperoxidase activity were determined. Peritoneal lavage fluid (PLF) was assayed for neutrophil infiltration and both aerobic and anaerobic bacterial counts. Results After intraperitoneal injection, cIAP activity in PLF decreased 50% within 15 min with minimal activity evident at 4 h. Compared with irrigation with normal saline, cIAP irrigation increased the 7-day survival rate in mice undergoing CLP, with maximal effects seen at 25 units of cIAP (0% vs. 46% survival rate, respectively; p < 0.001). cIAP treatment reduced lung inflammation, liver damage and levels of tumor necrosis factor alpha and interleukin-6. Conclusions Peritoneal irrigation with cIAP significantly enhances survival in a mouse model of peritonitis, likely through reduction of local inflammation and remote organ damage. We suggest that intraperitoneal cIAP irrigation could be a novel therapy for intra-abdominal sepsis.

Stress-induced analgesia was evaluated in adult rats submitted early in life to a protein deprivation schedule. Rats were undernourished with a hypoproteic diet containing 80 g casein/kg diet from d 14 of gestation until 50 days of age. Rats were thereafter fed a balanced nonpurified diet until 140 days of age, when they were exposed to two stressors: forced swimming and acute restraint, after which the analgesic response was evaluated. In addition, the analgesic response induced by different morphine doses was determined in another group of rats. Basal latency was not different in deprived and control rats. Undernourished rats presented a significantly lower analgesic response in both stress situations. However, when the analgesic response induced by different morphine doses (1, 2, 4 and 8 mg/kg, s.c.) was assessed, a significantly higher response occurred in undernourished rats compared to control rats. This lower stress-induced analgesia in undernourished rats may account for the behavioral alterations attributed to early undernutrition. J. Nutr. 127: 765-769, 1997. KEY WORDS: • perinatal undernutrition • opiate agent • rats • stress • analgesia • protein undernutrition 1 Supported by grants from the Consejo Nacional de Investigaciones Científicas y Té cnicas (CONICET) and from CONICOR, Có rdoba, Argentina.

Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one L-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure (C 120 ¼ 110.2873.69; BiLAO 120 ¼ 82.275.6 mmHg*); renal vascular resistance (C 120 ¼ 5.4870.53; BiLAO 120 ¼ 4.1270.42 mmHg/mL/g/min*), urinary flow (C 120 ¼ 0.16070.020; Bi-LAO 120 ¼ 0.06470.012 mL/g/min*), glomerular filtration rate (C 120 ¼ 0.69770.084; BiLAO 120 ¼ 0.17670.017 mL/g/min*), sodium (C 120 ¼ 79.7670.56; BiLAO 120 ¼ 65.3976.19%*), potassium (C 120 ¼ 69.9476.86; BiLAO 120 ¼ 60.2672.24%*) and chloride tubular reabsortion (C 120 ¼ 78.5372.33; BiLAO 120 ¼ 64.5876.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased ARTICLE IN PRESS www.elsevier.com/locate/toxicon $ Ethical statement: The experiments follow the methodology recommended by the international ethical standards of the scientific committee of our university (Comitteˆde È tica e Pesquisa com Animais). (H.S.A. Monteiro).

To provide re-description of Cryptosporidium cuniculus Inman and Takeuchi, 1979 (synonymous with rabbit genotype), a species closely related to Cryptosporidium hominis, the morphology, natural and experimental host specificity, and genetic characterisation were investigated. The morphology and diagnostic characteristics are typical of other intestinal species of Cryptosporidium, albeit with slightly larger oocysts (5.55-6.40 Â 5.02-5.92 lm; mean 5.98 Â 5.38 lm; length:width = 1.1; n = 50). Natural hosts appear to be European rabbits (Oryctolagus cuniculus) and humans (Homo sapiens). Experimental infections have been established in weanling rabbits (O. cuniculus), immunosuppressed Mongolian gerbils (Meriones unguiculatus) and immunosuppressed adult Porton strain mice (Mus musculus), but not in neonatal mice. Patterns of infection measured by oocyst shedding are significantly different compared with C. hominis, particularly in rabbits. Histological examination reveals endogenous stages in the brush border of the epithelium of the small intestinal villi, but clinical signs are absent. Inoculation of human HCT-8 cells results in discrete clusters of endogenous stages. A close relationship with C. hominis is inferred from molecular analyses at the ssrRNA, 70 kDa heat shock protein (HSP70), actin, Cryptosporidium oocyst wall protein (COWP), 60 kDa glycoprotein (GP60) genes and a region encoding a product of unknown function (LIB13). Sequences contained limited, consistent polymorphisms at the ssrRNA, HSP70 and actin genes, were identical at the COWP and LIB13 genes and demonstrated two unique families at the GP60 gene. Although genetically closely related, there are significant biological differences between C. cuniculus and C. hominis that support these protozoa being separate species. This is based on the current understanding of these organisms and relies on the assumption that mating between these species would not normally occur. If this is subsequently demonstrated their categorisation may need to be readdressed.

Harmaline inhibition of sodium uptake and of sodium-dependent o-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, "initial" o-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrations o-glucose uptake was stimulated by 10-15~. When a similar potassium gradient was used, harmaline had no effect. At concentrations upt to 2 mM, harmaline did not alter the equilibrium uptake of o-glucose or o-mannitol. After pre-equlibration with sodium (25 raM), o-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2raM. Sodium (10mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependent o-glucose uptake. Similar to phlorizin, harmaline (1 raM) was able to prevent glucose-induced sodium influx across the brush-border membrane.

Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocareinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border. Villin is one of the actin binding proteins found in microvilli which compose brush borders. Villin is expressed in the adult and in the embryo before the appearance of the brush border. Villin can be used as a tissue-specific marker for normal diffentiated and undifferentiated cells derived from gastrointestinal tractus in the adult as well as in the embryo. Since villin is a good marker for intestinal cells and plays a structural role in the assembly of the brush border we have analysed its expression and its localization in HT 29 cells. In HT 29 cells, as in the tissue, villin is synthesized at low levels before the appearance of the brush border. The high rate of synthesis and the recruitement of villin at the apical pole of the cells can be correlated with the existence of a well developed brush border.

Broilers chickens have limited ability to utilize phytate phosphorus (PP), and the influence of nutrients on the activity and efficacy of intestinal phytase is unclear. Therefore in a 2 × 2 × 2 factorial experiment, male chicks were fed 0 or 0.21 mg/kg 25-hydroxycholecalciferol (25-OH D 3 ), 4 or 9 g/kg Ca supplied from CaCO 3 or Ca malate from 14 to 24 d of age (six pens/treatment, eight birds/pen). Source of Ca had no effect on tibia ash, intestinal phytase activity, or apparent ileal PP hydrolysis. Phytase activity (V max ) within brush border vesicles prepared from small intestinal mucosa was greater in chicks fed 4 vs. 9 g/kg Ca (P ≤ 0.05). Similarly, birds fed 4 vs. 9 g/kg Ca were able to hydrolyze an additional 24.36% (Abbreviation Key: 25-OH D 3 = 25-hydroxycholecalciferol; BBV = brush border vesicles; ICP = inductively coupled plasma-emission spectroscopy; PP = phytate phosphorus; V max = maximum velocity of phytate hydrolysis. by guest on August 25, 2015 http://ps.oxfordjournals.org/ Downloaded from

Brush borders (microvilli) were isolated from Spodoptera frugiperda posterior midgut cells by using the cation differential precipitation technique. These microvilli preparations still contained some cytoskeleton elements visible by electron microscope. These elements are removed by Tris treatment followed by dilution and ultracentrifugation. Tris treatment solubilizes protein with M r values similar to some cytoskeleton proteins, and causes an increase in the specific activity of the marker enzyme (aminopeptidase) and in the lipid-protein ratio of the purified microvillar membrane. Microvilli preparations from anterior midgut cells seem to be free from cytoskeleton. This is supported by electron microscopy and by the absence, after Tris treatment, of changes in the specific activity of the marker enzyme and in the lipid-protein ratio. The cytoskeleton of these microvilli should assemble and disassemble to permit the passage of vesicles observed migrating through them. As a result, it is likely that the cytoskeleton is not rigid and is lost during microvilli preparation by differential precipitation. In comparison with other insects, S. frugiperda purified microvillar membranes have low cholesterol, carbohydrate and lipid-protein ratios. Because S. frugiperda midgut microvillar membranes almost lack intrinsic digestive enzymes, their low lipid-protein ratios might result from a large amount of carrier or other proteins.

The present study was undertaken in order to determine the effect of a dietary incorporation of soybean meal (SBM) on growth performance, nutrient utilization and gut histology of Egyptian sole Solea aegyptiaca. This species, which is genetically and morphologically close to Solea solea, is currently present in the Mediterranean Sea and reared in some Italian farms, using the same techniques for S. solea production and it could represent a promising new species for Mediterranean aquaculture.

Cubilin is a high molecular weight multiligand receptor that mediates intestinal absorption of intrinsic factor-cobalamin and selective protein reabsorption in renal tubules. The genetic basis of selective intestinal cobalamin malabsorption with proteinuria was investigated in a canine model closely resembling human Imerslund-Grä sbeck syndrome caused by cubilin mutations. Canine CUBN cDNA was cloned and sequenced, showing high identity with human and rat CUBN cDNAs. An intragenic CUBN marker was identified in the canine family and used to test the hypothesis of genetic linkage of the disease and CUBN loci. Linkage was rejected, indicating that the canine disorder resembling Imerslund-Grä sbeck syndrome is caused by defect of a gene product other than cubilin. These results imply that there may be locus heterogeneity among human kindreds with selective intestinal cobalamin malabsorption and proteinuria and that normal brush-border expression of cubilin requires the activity of an accessory protein.

The present work shows that semistarvation (8-10 g of food for 10 days) increases net D-glucose, Na § and water transport in the everted and perfused rat jejunum. A linear and positive correlation between cell sugar concentration and transport was found in control and semistarved rats, but the phenomenon was more relevant only in semistarved animals. Membrane vesicle experiments showed that semistarvation increases sugar overshoot only in brush border membrane vesicles, while this situation does not occur in basolateral membrane vesicles. The effect of partial food deprivation seems to enhance net sugar transport by increasing sugar entry across the apical membrane of enterocytes.

Pigs frequently encounter bacterial infections like enterotoxigenic Escherichia coli (ETEC). Due to rising concerns about antibiotic resistance of bacteria, there is a large demand for natural alternatives to combat these ETEC infections. Plant polyphenols have been suggested to reduce both the binding of cholera toxin to the GM1 ganglioside and the adhesion of uropathogenic E. coli to F1 fimbriae. In this study different commercial natural polyphenol extracts were evaluated for their possible effect on ETEC. Out of the three polyphenol extracts tested two exert inhibitory effects on the heat-labile toxin binding and all three extracts reduced the binding of ETEC to brush borders. The presence of protein abolished labile toxin binding properties of polyphenols. This makes it unlikely that these polyphenols could be used as an actual feed additive to combat ETEC caused diarrhea.

The principle of an enhanced gastrointestinal (GI) absorption of substances based on close contact with epithelial cells was used for the construction of a drug delivery system. The colonization of the small intestine by Escherichia coli strains is mediated by cell surface antigens called fimbriae, which enable bacteria to adhere to the brush border of epithelial cells. A special fimbriae antigen (K99) was isolated from an E. coli strain harbouring the fimbriae-encoding plasmid pRI9906. The functionality of isolated fimbriae was tested by a haemagglutination assay with equine erythrocytes, which represent the same K99-receptor-structures like Gl-epithelial cells. The adherence ability of an adhesive drug delivery system such as neutralized polyacrylic acid (PAA) could be improved by the covalent binding of K99-fimbriae. Affinity of the matrix-bound fimbriae to their receptor could be confirmed with equine erythrocytes, which migrate through a K99-PAA-gel ten times slower than through the same gel without fimbriae. Such a fimbriae-mediated bioadhesive drug delivery system could lead to enhanced drug absorption.

T-cell-mediated pathogenesis has been documented in various idiopathic and microbially induced intestinal disorders. Diffuse microvillous shortening seen in giardiasis is responsible for disaccharidase insufficiencies and malabsorption of electrolytes, nutrients, and water. Other mucosal changes include crypt hyperplasia and increased numbers of intraepithelial lymphocytes (IEL). A recent report using an athymic mouse model of infection showed that these epithelial injuries were dependent on T cells. The aim of the present study was to identify which subset of superior mesenteric lymph node (SMLN) T cells were responsible for mucosal alterations in giardiasis. CD4 ؉ and CD8 ؉ T cells, as well as whole lymphocyte populations, were isolated from SMLN of Giardia muris-infected mice for adoptive transfer. Jejunal segments of recipient mice were assessed for brush border ultrastructure, sucrase activity, crypt/villus ratio, and IEL numbers. Mice that received enriched CD8 ؉ and whole SMLN lymphocytes, but not CD4 ؉ T cells, from infected donors showed diffuse shortening of microvilli, loss of brush border surface area, impaired sucrase activity, and increased crypt/villus ratios compared to respective controls. Transfer of whole SMLN lymphocytes, as well as enriched CD4 ؉ or CD8 ؉ T cells, from infected donors led to increased IEL numbers in the recipient jejunum. The findings indicate that loss of intestinal brush border surface area, reduced disaccharidase activities, and increased crypt/villus ratios in giardiasis are mediated by CD8 ؉ T cells, whereas both CD8 ؉ and CD4 ؉ SMLN T cells regulate the influx of IEL.

Cow&amp;amp;amp;amp;amp;amp;amp;amp;#39;s milk stimulates growth in neonates and supplementation with specific milk proteins may benefit immuno-compromized neonates exhibiting poor growth. We investigated the effects of specific milk proteins (casein and whey) and plant protein (hydrolysed soy) on body and organ growth and bone mineralization in colostrum-deprived newborn pigs. Six days after birth, both casein and whey increased body and small intestinal

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH 2 -terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity = 21.6 units/mg and 17.5 units/µg, respectively; crude venom = 8.0 units/mg and 9.5 units/µg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala 14 -Leu 15 and Tyr 16 -Leu 17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 µg) subcutaneously into mice.

A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz = ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K m for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGF-SPFRQ-EDDnp was 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from www.elsevier.com/locate/ijbcb : S 1 3 5 7 -2 7 2 5 ( 0 0 ) 0 0 0 6 0 -1 B.M.R. Quinto et al. / 1162 kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.

Our current state of knowledge of the mechanism and regulation of intestinal iron absorption has been critically dependent on the analysis of inherited disorders of iron homeostasis in both humans and other animal species. Mutations in DMT1 and Ireg1 have revealed that these molecules are major mediators of iron transport across the brush border and basolateral membranes of the enterocyte, respectively. Similarly, the iron oxidase hephaestin has been shown to play an important role in basolateral iron efflux. The analysis of a range of human iron loading disorders has provided very strong evidence that the products of the HFE, TfR2, hepcidin and hemojuvelin genes comprise integral components of the machinery that regulates iron absorption and iron traffic around the body. Engineered mouse strains have already proved very effective in helping to dissect pathways of iron homeostasis, and in the future they will continue to provide important insights into the absorption of both inorganic and haem iron by the gut.

Brush border membrane vesicles (BBMV) purified from steer jejunum were used to study the kinetics of sodium Dglucose cotransport under voltage clamped, zero-trans conditions. When the initial rate of glucose transport (JgJuc) was measured over a wide ,range of glucose concentrations (IS] = 0.01-20 mM), curvature of the Woolf-Augustinsson-Hofstee plots was seen, compatible with a diffusional and one major, high capacity (maximal transport rate -/max = 5.8-8.8 nmol/mg 9 rain) saturable system. Further studies indicated that changes in cis [Na] altered the K,, but not the J .... suggesting the presence of a rapid-equilibrium, ordered bireactant system with sodium adding first. Trans sodium inhibited Jgtuc hyperbolically. KCl-valinomycin diffusion potentials, inner membrane face positive, lowered J~,r while potentials of the opposite polarity raise Jg~uc. At low glucose concentrations ([S] < 0.05 mM), a second, minor, high affinity transport system was indicated. Further evidence for this second saturable system was provided by" sodium activation curves, which were hyperbolic when [S] = 0.5 mM, but,were sigmoidal when IS] = 0.01 mM. Simultaneous fluxes of =Na and [3H]glucose at 1 mM glucose and 30 mM NaC1 yielded a cotransport-dependent flux ratio of 2 : 1 sodium/glucose, suggestive of 1 : 1 (Na/glucose) high capacity, low affinity system and a -3 : 1 (Na/glucose) high affinity, low capacity system. Kinetic experiments with rabbit jejunal brush borders revealed two major Nadependent saturable systems. Extravesicular (cis) Na changed the K,, but not the Jmax of the major system.

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

Folic acid deficiency is the most prevalent vitamin deficiency throughout the world and its effect on brush border membrane composition has not been studied earlier. We investigated the effect of folate deficiency on the structure and function of the intestinal brush border membrane. Various brush border enzyme activities, membrane sugars and lipids were evaluated in two groups of weanling male albino rats after 3 mo of feeding control and folate deficient diets. Except sucrase, all the other three enzymes, viz., alkaline phosphatase, leucine amino peptidase and ␥-glutamyl transpeptidase showed decrease in activity in rats fed folate-deficient diets. Among sugars, hexoses and hexosamines showed significant decline in amount whereas sialic acid content showed great increase in brush border membrane of folate-deficient rats as compared to controls. Furthermore, there was a significant reduction in cholesterol, phospholipids, triglycerides, cerebrosides and fucolipids in the group fed the folate-deficient diet. Our study suggests that folate deficiency results in altered enzyme activities, lipid and sugar composition of intestinal brush border membrane. Such changes might reflect the underlying cause of the gastrointestinal disturbances observed in folate deficiency.

Prostate-specific membrane antigen (PSMA) is a type II integral membrane glycoprotein that was initially characterized by the monoclonal antibody (mAb) 7E11. PSMA is highly expressed in prostate secretoryacinar epithelium and prostate cancer as well as in several extraprostatic tissues. Recent evidence suggests that PSMA is also expressed in tumorassociated neovasculature. We examined the immunohistochemical characteristics of 7E11 and those of four recently developed anti-PSMA mAbs (J591, J415, and Hybritech PEQ226.5 and PM2J004.5), each of which binds a distinct epitope of PSMA. Using the streptavidin-biotin method, we evaluated these mAbs in viable prostate cancer cell lines and various fresh-frozen benign and malignant tissue specimens. In the latter, we compared the localization of the anti-PSMA mAbs to that of the antiendothelial cell mAb CD34. With rare exceptions, all five anti-PSMA mAbs reacted strongly with the neovasculature of a wide spectrum of malignant neoplasms: conventional (clear cell) renal carcinoma (11 of 11 cases), transitional cell carcinoma of the urinary bladder (6 of 6 cases), testicular embryonal carcinoma (1 of 1 case), colonic adenocarcinoma (5 of 5 cases), neuroendocrine carcinoma (5 of 5 cases), glioblastoma multiforme (1 of 1 cases), malignant melanoma (5 of 5 cases), pancreatic duct carcinoma (4 of 4 cases), non-small cell lung carcinoma (5 of 5 cases), soft tissue sarcoma (5 of 6 cases), breast carcinoma (5 of 6 cases), and prostatic adenocarcinoma (2 of 12 cases). Localization of the anti-PSMA mAbs to tumor-associated neovasculature was confirmed by CD34 immunohistochemistry in sequential tissue sections. Normal vascular endothelium in non-cancer-bearing tissue was consistently PSMA negative. The anti-PSMA mAbs reacted with the neoplastic cells of prostatic adenocarcinoma (12 of 12 cases) but not with the neoplastic cells of any other tumor type, including those of benign and malignant vascular tumors (0 of 3 hemangiomas, 0 of 1 hemangioendothelioma, and 0 of 1 angiosarcoma). The mAbs to the extracellular PSMA domain (J591, J415, and Hybritech PEQ226.5) bound viable prostate cancer cells (LNCaP and PC3-PIP), whereas the mAbs to the intracellular domain (7E11 and Hybritech PM2J004.5) did not. All five anti-PSMA mAbs reacted with fresh-frozen benign prostate secretory-acinar epithelium (28 of 28 cases), duodenal columnar (brush border) epithelium (11 of 11 cases), proximal renal tubular epithelium (5 of 5 cases), colonic ganglion cells (1 of 12 cases), and benign breast epithelium (8 of 8 cases). A subset of skeletal muscle cells was positive with 7E11 (7 of 7 cases) and negative with the other four anti-PSMA mAbs. PSMA was consistently expressed in the neovasculature of a wide variety of malignant neoplasms and may be an effective target for mAb-based antineovasculature therapy.

Following preterm birth, bacterial colonization and enteral formula feeding predispose neonates to gut dysfunction and necrotizing enterocolitis (NEC), a serious gastrointestinal inflammatory disease. We hypothesized that administration of probiotics would beneficially influence early bacterial colonization, thereby reducing the susceptibility to formula-induced gut atrophy, dysfunction, and NEC. Caesarean-delivered preterm pigs were provided total parenteral nutrition (1.5 d) followed by enteral feeding (2 d) with porcine colostrum (COLOS; n = 5), formula (FORM; n = 9), or formula with probiotics (FORM-P; Bifidobacterium animalis and Lactobacillus: L. acidophilus, L. casei, L. pentosus, L. plantarum; n = 13). Clinical NEC scores were reduced (P &lt; 0.05) in FORM-P (2.0 +/- 0.2) and COLOS groups (1.7 +/- 0.5) compared with FORM pigs (3.4 +/- 0.6). Lower NEC scores were associated with elevated intestinal weight, mucosa proportion, villus height, RNA integrity, and brush border amin...

Morphological and functional changes were examined in the upper jejunum and terminal ileum of 18 patients suffering from Crohn's disease. Intestinal permeability, biochemical determination of enzymatic activities, and morphologic evaluation of the severity of the lesions were evaluated. Ulcerative colitis and irritable bowel syndrome patients served as controls. We found abnormal lactulose-mannitol tests in all patients with active Crohn's disease. Permeability changes correlated with increased crypt cell proliferation, as indicated by thymidine kinase activity. A significant reduction in brush border enzyme activities was seen in the terminal ileum, but no significant change was observed in the unaffected upper jejunum. The number of mast cells was increased in the diseased ileum. We conclude that the site of inflammation and the healing capacity of the epithelium are important in determining functional and biochemical abnormalities in active Crohn's disease. Changes may be dependent on the type and number of immune cells involved in the inflammatory process.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 L L-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k cat / K m ) for the hydrolysis of quercetin-4P P-glucoside, quercetin-3glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM 31 s 31 ) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal E. coli during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that C. rodentium colonizes the rectum, a site previously unreported for this pathogen.

Background The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level. Methods Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses. Results We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins. Conclusion We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function.

Experimental model of lead nephropathy. I. Continuous high.dose lead administration. This study followed the progression of lead nephropathy in male Sprague-Dawley rats (E) administered lead acetate (0.5%) continuously in drinking water for periods ranging from Ito 12 months. Control animals (C) were pair-fed. Observations included renal pathology by light and electron microscopy, Wet and dry kidney weights, and glomerular filtration rate (GFR) to assess renal function. Urinary excretion of lead, the enzymes N-acetyl-beta-D-glucosaminidase (NAG) and glutathione-S-transferase (GST), and brush border antigens (BB5O, CG9, and HF5) were utilized to explore possible markers of kidney injury. GFR was increased significantly after three months of lead exposure, but was decreased significantly after 12 months. Kidney wet weights were significantly greater in E than C from three months on. Kidney dry weight/wet weight ratio was constant up to three months, but decreased in E at 12 months. Glomerular diameters were normal at all time periods; the nephromegaly was related primarily to hypertrophy of proximal tubules. Lead inclusion bodies were found in nuclei of proximal convoluted tubules and pars recta at all times.

cDNA encoding CYCLE (CYC) from the coleseed sawfly, Athalia rosae (Hymenoptera, Symphyta), was amplified by PCR. This is a first determination of hymenopteran CYC structure. ArCYC had an overall identity of 66% with CYC of Anopheles gambiae and ca. 60% of Drosophila melanogaster. Structural investigation revealed that ArCYC contained characteristic motifs of: bHLH, PAS A, PAS B, PAC and BCTR. Detailed analysis indicated high conservation of these regions among insects. Northern blot analysis showed that the mRNA of ca. 3 kb was transcribed both in the head and in the rest of the body. Southern blot analysis suggested the presence of a single copy of the gene in the genome. Western blot indicates that the quantity of CYC protein does not fluctuate under LD 12:12 in either the head or the rest of the body. Immunocytochemical examination revealed CYC-like antigen in the pars intercerebralis, dorsolateral protocerebrum, dorsal optic tract, tritocerebrum of the brain and the subesophagea...